Identification of gallbladder cancer by direct near-infrared measurement of raw bile combined with two-trace two-dimensional correlation analysis.

We demonstrated the utility of direct near-infrared (NIR) bile analysis for the identification of gallbladder (GB) cancer by employing two-trace two-dimensional (2T2D) correlation analysis to recognize dissimilar spectral features among diverse bile samples for potential improvement of discrimination accuracy. To represent more diverse clinical cases for reliable assessment, bile samples obtained from five normal, 44 gallstone, 25 GB polyp, six hepatocellular cancer (HCC), and eight GB cancer subjects were analyzed. Due to the altered metabolic pathways by carcinogenesis, the NIR spectral features of GB cancer samples, including intensity ratios of main peaks, were different from those of other sample groups. The differentiation of GB cancer in the principal component (PC) score domain was mediocre and subsequent discrimination accuracy based on linear discriminant analysis (LDA) was 88.5%. When 2T2D slice spectra were obtained using a reference spectrum constructed by the linear combination of the spectra of five pure representative bile metabolites and employed, the accuracy was improved to 95.6%. The sensitive recognition of dissimilar spectral features in GB cancer by 2T2D correlation analysis was responsible for the enhanced discrimination.

Feasibility of Raman spectroscopic identification of gall bladder cancer using extracellular vesicles extracted from bile.

Extracellular vesicles (EVs), which are heterogeneous membrane-based vesicles with bilayer cell membrane structures, could be versatile biomarkers for the identification of diverse diseases including cancers. With this potential, this study has attempted the Raman spectroscopic identification of gall bladder (GB) cancer by directly measuring the EV solution extracted from human bile without further sample drying. For this purpose, bile samples were obtained from four normal individuals and 21 GB polyp, eight hepatocellular carcinoma (HCC), and five GB cancer patients, and EVs were extracted from each of the bile samples. The Raman peak shapes of the EVs extracted from the GB cancer samples, especially the relative intensities of peaks in the 1560-1340 cm-1 range, were dissimilar to those of the samples from the normal, GB polyp, and HCC groups. The intensity ratios of peaks at 1537 and 1453 cm-1 and at 1395 and 1359 cm-1 of the GB cancer samples were lower and higher, respectively, than those of the samples of the remaining three groups. The differences of peak intensity ratios were statistically significant based on the Mann-Whitney U test. DNA/RNA bases, amino acids, and bile salts contributed to the spectra of EVs, and their relative abundances seemed to vary according to the occurrence of GB cancer. The varied metabolite compositions and/or structures of EVs were successfully demonstrated by the dissimilar peak intensity ratios in the Raman spectra, thereby enabling the discrimination of GB cancer.

Combination of LIBS-based elemental analysis and near-infrared molecular fingerprinting of bile juice to enhance identification of gallbladder cancer.

Laser-induced breakdown spectroscopy (LIBS) and near-infrared (NIR) spectroscopy, enabling the measurement of raw bile directly without sample pretreatment, were cooperatively combined to enhance the discrimination of gallbladder cancer (GBC) from other diseases of gallstone and gallbladder (GB) polyp. Since elemental contents and metabolite compositions of bile vary according to the pathological conditions of pancreaticobiliary patients, the use of complementary information could be synergetic to improve disease identification accuracy. The ratios of Mg and Na peak areas (AMg/ANa) and Na and K peak areas (ANa/AK) in the LIBS spectra of GBC samples were different from those of the remaining samples. Also, the intensity ratios of main NIR peaks differed in GBC. Nonetheless, the use of only element peak area ratio or NIR peak intensity ratio was not sufficient to clearly discriminate GBC. On the other hand, when the ANa/AK values and second NIR principal component scores were combined, the discrimination of GBC from normal/gallstone/GB polyp was substantially enhanced owing to incorporation of both complementary GBC-discriminant spectroscopic signatures.

Feasibility of discrimination of gall bladder (GB) stone and GB polyp using voltage-applied SERS measurement of bile juice samples in conjunction with two-trace two-dimensional (2T2D) correlation analysis.

Voltage-applied SERS measurement of bile juice in conjunction with two-trace two-dimensional (2T2D) correlation analysis was demonstrated as a potential tool to enhance discrimination of gall bladder (GB) stone and GB polyp. When SERS spectra of the aqueous phases extracted from raw bile juice samples were measured with applying external voltage from -300 to +300 mV (100 mV intervals), subsequent spectral variations of the adsorbed components (bilirubin-containing compounds) on the SERS substrate were minute, and discrimination of the two GB diseases in a principal component score domain was difficult. Therefore, 2T2D correlation analysis, effectively identifying asynchronous (dissimilar) spectral behaviors in the voltage-induced SERS spectra, was used to improve the discrimination. When two spectra of a sample collected with application of +100 and +300 mV were adopted, the features of subsequent 2T2D slice spectra were characteristic, and discrimination of GB stone and GB polyp substantially improved. External voltage application and recognition of the voltage-induced spectral features by 2T2D correlation analysis were key factors for the improvement. Since the demonstrated method relied on only a few SERS-active compounds, infrared (IR) spectroscopy featuring all the present components in the samples was also evaluated for comparison. However, the IR-based discrimination was inferior because the metabolite compositions in the samples between the GB diseases were not noticeably different.

Spatially offset Raman scattering line-mapping as a potential tool for particle size analysis.

A spatially offset Raman spectroscopy (SORS) line-mapping scheme was explored as a tool for the measurement of particle size. The proposed scheme is based on the fact that photon migration in powder packing varies as a function of the reduced scattering coefficient, which is directly related to the particle size of the sample. It is known that a smaller particle yields a larger reduced scattering coefficient. Therefore, recognition of the particle size-dependent photon migration (distribution) could be a means to determine the sample's particle size and SORS is a versatile tool for this purpose. Peak intensities acquired along the SORS mapping line are expected to decrease with an increase of the offset distance and the descending slope of the peak intensity can be translated into particle size, for example, a greater slope (steeper intensity decrease) for smaller particles yielding a narrower (denser) photon distribution. For the study, low-density polyethylene (LDPE) and middle-density PE (MDPE) powders with four particle sizes were measured. In each case, the slope of intensity decrease became less steep with the increase of particle size due to the broader photon distribution. A comparative analysis of LDPE and MDPE spectra found that the slope was steeper in the measurement of MDPE powder since the photon distribution was narrower owing to the high particle density. Together, these findings suggest that the proposed scheme is potentially expandable to measure particle sizes of samples with relevant prior calibration and provide useful information on sample composition also for chemical analysis.

Use of Multiple Bacteriophage-Based Structural Color Sensors to Improve Accuracy for Discrimination of Geographical Origins of Agricultural Products.

A single M13 bacteriophage color sensor was previously utilized for discriminating the geographical origins of agricultural products (garlic, onion, and perilla). The resulting discrimination accuracy was acceptable, ranging from 88.6% to 94.0%. To improve the accuracy further, the use of three separate M13 bacteriophage color sensors containing different amino acid residues providing unique individual color changes (Wild sensor: glutamic acid (E)-glycine (G)-aspartic acid (D), WHW sensor: tryptophan (W)-histidine (H)-tryptophan (W), 4E sensor: four repeating glutamic acids (E)) was proposed. This study was driven by the possibility of enhancing sample discrimination by combining mutually characteristic and complimentary RGB signals obtained from each color sensor, which resulted from dissimilar interactions of sample odors with the employed color sensors. When each color sensor was used individually, the discrimination accuracy based on support vector machine (SVM) ranged from 91.8-94.0%, 88.6-90.3%, and 89.8-92.1% for garlic, onion, and perilla samples, respectively. Accuracy improved to 98.0%, 97.5%, and 97.1%, respectively, by integrating all of the RGB signals acquired from the three color sensors. Therefore, the proposed strategy was effective for improving sample discriminability. To further examine the dissimilar responses of each color sensor to odor molecules, typical odor components in the samples (allyl disulfide, allyl methyl disulfide, and perillaldehyde) were measured using each color sensor, and differences in RGB signals were analyzed.

Incorporating Non-NIR Absorbing Agent into Packed Powder Samples in Diffuse Reflectance NIR Measurement to Improve Representation of Sample Composition and Accuracy of Concentration Determination.

A strategy to improve accuracy of near-infrared (NIR) quantitative analysis of powder samples by alleviating the subsampling problem is demonstrated. The approach is to increase spectroscopic sampling volume for more accurate representation of its composition by filling the void space in a packed sample with liquid to reduce differences in refractive index between particle and unoccupied space. By this way, NIR radiation can propagate deeper into the packed powder due to decreased degree of reflection at the interfaces, leading to greater sampling volume. Perfluorohexane (PFH) was chosen as the space-filling liquid since it does not readily absorb NIR radiation and does not disintegrate sample particles due to its strong hydrophobicity. For evaluation, binary mixtures composed of ambroxol and lactose at different concentrations were prepared, and their diffuse reflectance spectra were collected in two ways: conventional direct powder measurement and PFH-incorporated powder measurement. The accuracy of ambroxol concentration determination improved in the PFH-incorporated measurement due to acquisition of more composition-representative spectra by the greater sampling volume. Also, the sample morphology did not change in PFH medium based on XRD analysis. In parallel, Monte Carlo simulation was executed to track NIR photons in the samples and explain the improved accuracy. Improvement in accuracy was also realized when analyzing real pharmaceutical samples containing an active pharmaceutical ingredient in granular form, demonstrating expandability of the proposed scheme.

Feasibility of Voltage-Applied SERS Measurement of Bile Juice as an Effective Analytical Scheme to Enhance Discrimination between Gall Bladder (GB) Polyp and GB Cancer.

A unique surface-enhanced Raman scattering (SERS) measurement scheme to discriminate gall bladder (GB) polyp and GB cancer by analysis of bile juice is proposed. Along with the high sensitivity of SERS, external voltage application during SERS measurement was incorporated to improve sample discriminability. For this purpose, Au nanodendrites were constructed on a screen-printed electrode (referred to as AuND@SPE), and Raman spectra of extracted aqueous phases from raw bile juice samples were acquired using the AuND@SPE at voltages from -300 to 300 mV. The sample spectra resembled that of bilirubin, possessing an open chain tetrapyrrole, showing that bilirubin derivatives in bile juice were mainly responsible for the observed peaks. Discrimination of GB polyp and GB cancer using just the normal SERS spectra was not achieved but became apparent when the spectra were acquired at a voltage of -100 mV. When voltage-applied SERS spectra of bilirubin and urobilinogen (one of bilirubin's derivatives) were examined, a sudden intensity elevation occurring at -100 mV was observed for urobilinogen but not bilirubin. Based on examination of corresponding cyclic voltammograms, the potential-driven strong adsorption of urobilinogen (no faradaic charge transfer) on AuND occurring at -100 mV induced a substantial increase in SERS intensity. It was presumed that the content of urobilinogen in the bile juice of a GB cancer patient would be higher than that of a GB polyp patient, and the contained urobilinogen was sensitively highlighted by applying -100 mV during SERS measurement, allowing clear discrimination of GB cancer against GB polyp.

Influence of interfering co-appearing container peaks on the accuracy of direct quantitative Raman measurement of a sample in a plastic container.

The axially perpendicular offset (APO) scheme was previously demonstrated as a versatile scheme able to minimize or eliminate the glass background in the direct and non-sampling Raman measurement of an ethanol sample housed in a glass bottle. Alternatively, when directly analyzing a sample housed in a plastic container, another typical container yielding strong Raman peaks itself, the Raman peaks of both the container and the housed sample are unavoidably present together in a collected spectrum. Therefore, a crucial issue to investigate under this situation is how the magnitude of the co-appearing container peaks influences the accuracy for quantitative analysis of the housed sample. For the evaluation, a non-sampling Raman spectroscopic measurement of the urea concentration in a urea gel housed in a circular polypropylene (PP) container was attempted by employing two axially perpendicular offset (APO) schemes with detection windows of different sizes (25.4 and 10.0 mm, referred to as the wide-window APO (WW-APO) and narrow-window APO (NW-APO), respectively), and transmission and back-scattering schemes incorporating a 25.4 mm detection window. The intensity ratios between the container and urea peaks in the collected spectra were different depending on the adopted measurement scheme. The intensity ratio was greatest (smallest container peak) in the NW-APO measurement due to the narrowed detection window, making the generated container Raman photons at the side-wall less detectable to the bottom-positioned detector. A spectral acquisition scheme allowing the maximal suppression of the container peaks, while still maintaining the sample features, was a key requirement to secure an accurate measurement of the sample concentration. In addition, a Monte Carlo simulation was used to visualize the distributions of the container and urea photons inside the sample-housed container.

Hyperspectral Raman Line Mapping as an Effective Tool To Monitor the Coating Thickness of Pharmaceutical Tablets.

Protective chemical coatings are deposited on drugs during the manufacturing process for the purpose of controlling the pharmacokinetics of active pharmaceutical ingredients (APIs). Although manufacturers attempt to coat all the tablets uniformly, the film thickness of an individual drug is statistically different and depends on the measuring position of the anisotropic structure, and analytical methods for measuring coating thickness must be robust to statistical and geometrical aberrations. Herein, we demonstrate that a spatially offset Raman-spectroscopy-based line mapping method offered excellent calibration and prediction of the coating thickness of 270 acetaminophen ( N-acetyl-para-aminophenol, paracetamol) tablets. Raman-scattered light resurfaced back from the coating and APIs, and offset-resolved spectra were projected according to the vertical positions in an imaging sensor. The Raman intensity ratio between the coating substance and the inner APIs is a key parameter in the analysis, and its variation with respect to the spatial offset is proportional to the coating thickness and duration. The results of this study have implications for the rapid spectroscopic thickness measurement of industrial products coated with transparent or translucent materials.

A sample wetting strategy to overcome differences in physical morphology between lab-prepared training samples and pharmaceutical process samples for reliable quantitative Raman spectroscopic analysis.

As a training set for multivariate quantitative analysis, lab-blended powder samples are frequently used due to the facile concentration variation of the individual components. Real pharmaceutical process samples, however, are often granular in form. The different morphologies between training and process samples result in dissimilar Raman spectral features, thereby seriously deteriorating the accuracy of the analysis. To overcome this hurdle, an effective and simple strategy to make physical presentations of both training and process samples similar, called water wetting, was demonstrated in this study. Wetting potentially dismantles process granule samples into smaller aggregates, whereas lab powder samples could be somewhat aggregated by water-bridging. Thus, water wetting would produce similar morphologies of both samples. To evaluate the strategy, samples containing esomeprazole magnesium dihydrate (EMD) as an active pharmaceutical ingredient (API) and five additional components were employed. Initially, training samples were mixed with the individual components at the intended concentration ranges. A partial least squares (PLS) model developed using the Raman spectra of the training samples was inaccurate in determining EMD concentrations in the real process samples. When both training and process samples were wetted prior to analysis, accurate concentration determination was realized due to no significant difference in Raman spectral features between samples. The similar morphologies and crystallinities of the two types of wetted samples were confirmed by analyzing the corresponding SEM images and XRD patterns. The proposed strategy is versatile and expandable for further vibrational spectroscopic analysis of various other samples with different morphologies.

Feasibility study for rapid near-infrared spectroscopic identification of different gallbladder diseases by direct analysis of bile juice.

A whole-sample-covering near-infrared (NIR) spectroscopy scheme has been adopted for the simple drop-and-dry measurement of raw bile juice for the identification of gallbladder (GB) diseases of stone, polyp, and cancer. For reproducible measurement, a non-NIR absorbing polytetrafluoroethylene (PTFE) providing a hydrophobic surface was chosen as a substrate to form bile juice droplets of a consistent shape. To ensure representative spectroscopic sampling, NIR radiation illuminated the whole area of the dried sample for spectral acquisition. The NIR band shapes and relative band intensities of GB cancer differed moderately from those of GB stone and GB polyp. The composition of GB cancer samples was presumed to be dissimilar from other sample compositions. Differentiation between GB polyp and GB stone, however, was less facile; nevertheless, in the case of GB polyp samples, the obtained NIR features were informative in the identification of various pathological conditions such as adenomyomatosis (abnormal growth of epidermal tissue) and hepatitis B. To elucidate the NIR features of bile juice samples, separate NIR spectra of major bile constituents such as conjugated bile salts, lecithin, cholesterol, and albumin were analyzed. The demonstrated NIR spectroscopy scheme requiring no sample pretreatment or separation of bile juice could be useful for fast bile juice-based screening of GB diseases, especially the identification of early GB cancer.

Improved infrared spectroscopic discrimination between gall bladder (GB) polyps and GB cancer using component-descriptive spectral features of separated phases from bile.

This study demonstrates a unique strategy for enhancing infrared (IR) spectroscopic discrimination between gall bladder (GB) polyps and cancer. This strategy includes the separation of raw bile juice into three sections of organic, aqueous, and amphiphilic phases and a cooperative combination of all IR spectral features of each separated phase for the discrimination. Raw bile juice is viscous and complex in composition because it contains fatty acids, cholesterol, proteins, phospholipids, bilirubin, and other components; therefore, the acquisition of IR spectra providing more component-discernible information is fundamental for improving discrimination. For this purpose, raw bile juice was separated into an aqueous phase, mostly containing bile salts, an organic phase with isolated lipids, and an amphiphilic phase, mainly containing proteins. The subsequent IR spectra of each separated phase were mutually characteristic and complementary to each other. When all the IR spectral features were combined, the discrimination was improved compared to that using the spectra of raw bile juice with no separation. The cooperative integration of more component-specific spectra obtained from each separated phase enhanced the discrimination. In addition, the IR spectra of the major constituents in bile juice, such as bile acids, conjugated bile salts, lecithin, and cholesterol, were recorded to explain the IR features of each separated phase.

Discrimination of phthalate species using a simple phage-based colorimetric sensor in conjunction with hierarchical support vector machine.

Here, the analytical potential of an M13 bacteriophage-based color sensor for discrimination of 4 phthalates with similar molecular structures (bis-(2-ethylhexyl)-phthalate (BEHP), dibutyl phthalate (DBP), diethyl phthalate (DEP), and benzyl-butyl-phthalate (BBP)) was investigated. The pattern and magnitude of the RGB color changes were different depending on the functional groups present in the phthalate structures. For example, BEHP possessing a long alkyl chain resulted in a minute color change, while the variation of color was substantially large when BBP containing an additional benzene ring was measured. Since a tryptophan-histidine-tryptophan residue possessing indole and imidazole was present on the self-assembled phages, the π-π interaction of benzene in BBP with the sensor surface produced a considerably greater color change. To evaluate the multi-modally varying color signals due to diverse interactions of the phthalates with the sensor and to discriminate them, support vector machine (SVM), which can construct a boundary hyperplane among complexly scattered sample groups, was used. In addition, hierarchical SVM (H-SVM) was adopted to deal with multi-class discrimination. The use of H-SVM improved the discrimination accuracy up to 90.1%, compared to 87.1% using SVM. The demonstrated color sensor is versatile and can be potentially adopted as an on-site screening tool. Strategies to improve the accuracy further for real applications are also discussed.

Axially perpendicular offset scheme for obtaining Raman spectra of housed samples in glass bottles with minimized glass-peak background.

An axially perpendicular offset (APO) scheme based on an axially perpendicular geometrical arrangement of laser illumination and photon detection, enabling spatially offset Raman spectroscopy (SORS), is proposed as a versatile tool for the minimization of the glass background in direct measurements of Raman spectra of samples housed in glass bottles. This strategy is based on the possibility of isolating glass photons from sample photons by properly locating a detector beneath the sample-housing bottle, because glass photons are much more localized near the glass wall while sample photons are widely distributed throughout the bottle. In addition, the curved bottom of the glass vial enabling forming the conical photon-detection volume would be further effective in exclusion of the glass photons in the acquisition of sample spectra. The APO scheme was validated by measuring the Raman spectra of 66% ethanol housed in four glass bottles of different sizes and colors; the measurements were performed by varying the offset distance from 2 mm to 20 mm. The intensity of the glass background decreased rapidly with increasing the offset distance; on the other hand, the variation in the ethanol intensity was relatively insignificant. In all cases, the offset distance of 16 mm minimized the presence of glass background in the spectra, thereby helping to highlight the pure ethanol bands and producing nearly similar sample spectral features regardless of contained bottles. The results of the Monte Carlo simulation were in accordance with the experimental observations, and the suppression of glass photons in the APO scheme was clearly explained and visualized by the simulation.

Investigation of the particle size-dependent near-infrared spectral features of binary mixture samples in conjunction with Monte Carlo simulation and the influence of particle size on the accuracy of quantitative analysis.

Near-infrared (NIR) spectral features of a target component can be influenced by its particle size as well as the particle sizes of the surrounding components in multi-component samples; therefore, understanding the essence of particle size-induced spectral variation is fundamental for robust quantitative analysis. For systematic investigation, two different types of binary-component samples were prepared. First, in ambroxol/lactose powders, only the particle size of lactose (surrounding filler) was varied, while the particle size of ambroxol (a target component) remained unchanged. Second, in lactose/PE pellets, the particle size of lactose (a target component) was only varied. When measuring ambroxol/lactose powders, the absorbances of both ambroxol and lactose peaks were elevated as the particle size of lactose increased. The larger lactose particle made the photon propagation broader in the sample, so a greater amount of ambroxol was able to interact with NIR photons; this led to an increase in absorbance. In the case of lactose (10 wt%)/PE pellets, the absorbance of lactose oppositely decreased as the lactose particle size increased. Since PE was the dominant component (85-95%) and had a small particle size (7 µm), the depth of photon propagation was substantially shallow. In this situation, the larger lactose particles would sometimes not be located within the small sampling volume and/or the photons would only partially interact with the particles; this led to a decrease in the absorbance. For both binary samples, the accuracies for the determination of target concentrations deteriorated as the particle size increased. This was due to the degradation of the measurement reproducibility caused by the increased uncertainty in the photon propagation. Monte Carlo simulation was effective for probing the photon propagation inside samples and supportive to explain the experimental observations.

Fast and non-destructive Raman spectroscopic determination of multi-walled carbon nanotube (MWCNT) contents in MWCNT/polydimethylsiloxane composites.

A versatile Raman spectroscopic method to determine the contents of carbon nanotubes (CNTs) in CNT/polydimethylsiloxane (PDMS) composites is demonstrated, and important issues directly related to the accuracy of the measurement have been investigated. Initially, Raman microscopic mappings over an area of 6.0 × 6.0 mm2 were carried out on CNT/PDMS composites, which revealed the existence of the partial localization of CNTs on a microscopic scale. Therefore, a laser illumination scheme covering a large sample area of 28.3 mm2 was employed to acquire a sample spectrum representative of the whole CNT concentration. The peak area ratio between the CNT and PDMS peaks clearly varied with the CNT concentration, whereas the reproducibility of measurements was degraded for the composites containing more than 3.0 wt% CNTs because of the decreased Raman sampling volume arising from the absorption of laser radiation by the CNTs. The laser penetration depth was semi-quantitatively investigated by observing the spectra of thin-sliced samples collected by positioning a Teflon disk behind the sample, and Monte Carlo simulations were employed to examine the internal photon propagation as well as explain the experimental observation. In summary, the fundamental issues affecting the Raman measurement of the CNT containing polymer matrix have been clearly addressed, and the finding here will be a beneficial basis for successful Raman spectroscopic analysis of different CNT-containing composites.

Characterization of Raman Scattering in Solid Samples with Different Particle Sizes and Elucidation on the Trends of Particle Size-Dependent Intensity Variations in Relation to Changes in the Sizes of Laser Illumination and Detection Area.

We have systematically characterized Raman scatterings in solid samples with different particle sizes and investigated subsequent trends of particle size-induced intensity variations. For this purpose, both lactose powders and pellets composed of five different particle sizes were prepared. Uniquely in this study, three spectral acquisition schemes with different sizes of laser illuminations and detection windows were employed for the evaluation, since it was expected that the experimental configuration would be another factor potentially influencing the intensity of the lactose peak, along with the particle size itself. In both samples, the distribution of Raman photons became broader with the increase in particle size, as the mean free path of laser photons, the average photon travel distance between consecutive scattering locations, became longer under this situation. When the particle size was the same, the Raman photon distribution was narrower in the pellets since the individual particles were more densely packed in a given volume (the shorter mean free path). When the size of the detection window was small, the number of photons reaching the detector decreased as the photon distribution was larger. Meanwhile, a large-window detector was able to collect the widely distributed Raman photons more effectively; therefore, the trends of intensity change with the variation in particle size were dissimilar depending on the employed spectral acquisition schemes. Overall, the Monte Carlo simulation was effective at probing the photon distribution inside the samples and helped to support the experimental observations.

Use of Multiple Peptide-Based SERS Probes Binding to Different Epitopes on a Protein Biomarker To Improve Detection Sensitivity.

We propose an analytical strategy to improve the sensitivity for detecting a protein biomarker through signal multiplication by manipulating multiple peptide-based surface-enhanced Raman scattering (SERS) probes to bind the biomarker. Protective antigen (PA) was used as an Anthrax biomarker in this study. For this purpose, five small peptides selective to various PA epitopes with different binding affinities were chosen and peptide-conjugated Au nanoparticle (AuNP) SERS probes were individually prepared using each peptide. Initially, five different SERS probes were separately used to detect PA and the sensitivities were compared. Next, the possibility of enhancing sensitivity by employing multiple SERS probes was examined. Rather than applying the probes simultaneously, which would induce competitive binding, each probe was added sequentially and an optimal probe-addition sequence was determined to provide maximal sensitivity. Finally, PA samples at seven different concentrations were measured with the optimal sequence. The limit of detection (LOD) was 0.1 aM, and the enhancement was more effective at lower PA concentrations. The proposed scheme can be further applicable to detect other protein biomarkers to diagnose various diseases.

Sensitive Surface Enhanced Raman Scattering-Based Detection of a BIGH3 Point Mutation Associated with Avellino Corneal Dystrophy.

Surface enhanced Raman scattering (SERS) is highly useful for sensitive analytical sensing; however, its practical availability for detecting a point mutation associated with disease in clinical sample was rarely proved. Herein, we present a toehold-mediated, DNA displacement-based, SERS sensor for detecting point mutations in the BIGH3 gene associated with the most common corneal dystrophies (CDs) in a clinical setting. To diagnose Avellino corneal dystrophy (ACD), selectivity was ensured by exploring optimal DNA displacement conditions such as length of toehold and hybridization temperature. A SERS-efficient Ag@Au bimetallic nanodendrite was employed to ensure sensitivity. Optimization for a clinical setting showed that discrimination was maximized when toehold length was 6-mer (T6), and hybridization temperature was 36 °C. On the basis of tests that used clinical homozygous and heterozygous CD samples, a single-base mismatched DNA sequence was identifiable within 30 min with a limit of detection (LOD) of 400 fM. From the results, we conclude that our toehold-mediated, DNA displacement-based, SERS sensor allows a rapid and sensitive detection of a BIGH3 gene point mutation associated with Avellino corneal dystrophy, indicating the practical ability of the method to diagnose genetic diseases caused by point mutations.